Application fields of ddPCR

Introduction

Droplet digital Polymerase Chain Reaction (ddPCR) is a new upgraded type of Polymerase
Chain Reaction (PCR) with a novel characteristic that can multiply and quantify target
nucleic acids highly precisely. ddPCR, a third-generation PCR, is a molecular tool for DNA
identification from any source which applies microfluidic technology and emulsion
chemistry (1-3).

The identification and diagnosis of biomarkers are crucial for any investigation type related
to nucleic acids; this is why it is indispensable to use biomolecular tools such as ddPCR to
analyze genetic material and find unusual features (4). This biomolecular tool applies to a
wide variety of search fields. However, there are four basic applications of ddPCR:
absolute quantification, copy number variation, rare event detection, and gene expression
(2).

This scientific review aims to supply an overview of ddPCR function, differences with
others PCRs and applications.

Results

ddPCR

ddPCR is an improved molecular technique that supplies information about target genetic
material with high precision, enhanced sensitivity and absolute quantification. This
technique can be applied in unlimited fields such as human, animal, plants and
microorganisms (5) as shown in table 2. This approach amplifies genetic material into
millions of copies, creating about 20 thousand nanoliter droplets to find biomarkers (6).
Droplets contain many, a few, or none of the DNA copies, and this is because DNA
molecules are distributed randomly in the recipients. Furthermore, droplets are the place
where ddPCR reactions take place. Finally, a special machine uses a fluorescence detector
to analyze these droplets, and a Poisson modeling equation is used to quantify the whole
copy numbers of the target gene material. (7) the whole process is shown in figure 1.

ddPCR and other PCRs

Conventional PCR is highly useful for analyzing and amplifying DNA sequences.
However, the variety and the huge or small quantity of DNA found in a sample requires
novel technology as ddPCR has (8). ddPCR supplies better precise, direct quantification
without needing a standard curve, but other PCRs obligatorily need a calibration curve to
function (9). Even ddPCR can cover study fields that other PCRs are unable; examples of
this are explained in table 2. ddPCR has notable advantages over other types of PCRs, as it
showed in table 1.
ddPCR application

Absolute quantification

ddPCR works without a house-keeping gene when counting the concentration of target
nucleic acid sequences in each sample, making this approach accurate for target DNA
measurements: ddPCR supplies a concentration of target nucleic acids copies per
microliter for a given sample without needing to run standard curves, making this approach
accurate for target sequences measurements included bacteria, parasites, and viruses (10),
viral load analysis such as in HIV DNA exact quantification in which ddPCR provides the
most accurate measurement of virus genetic material in patients infected (11); and
microbial quantification as it was probed in an essay in which a mixture with different
bacteria strains was quantified and identified using direct droplet digital PCR (dddPCR)
(12).

Copy number variation

Gene copy number variation (GCV) means alterations in a DNA target sequence, the genes
affected by this alteration are responsible for phenotypic changes, complicated behavioral
characteristics, and illness. ddPCR allows developing an efficient measurement of
variances of gene copy number (2). Even in equal species, such as humans or monkeys,
thousands of genomic sequences vary in copy number from one to another; therefore,
duplications or deletions of nucleotides can cause these alterations in a particular locus
concerning a reference locus within a cell (13). However, the physiopathology of these
alterations is still unclear, such as gene expression, protein functions, and, eventually,
disease. Therefore, the application of ddPCR for this purpose is wide; researchers have
used it to analyze GNC in microorganisms, food, and even cancer cells (2, 14).

Rare event detection

The rare variant refers to a single nucleotide polymorphism (SNP) or a translocation. The
rare event detection process quantifies DNA target sequences and can accurately detect rare
mutants as low as 0.0005% and rare sequences as low as 0.00013%. Single genes in a
sample must be amplified, such as nucleic acids from tumor cells, plants, pathogens, or
animals. ddPCR is highly sensitive to detecting rare mutations or sequences (2, 5). There
are two subtypes of this approach: rare mutation detection and rare sequence detection.

Gene expression

Gene expression (GE) and microRNA analysis applying ddPCR supplies complete
quantification of expression levels, even in low genetic material quantity as DNA or
microRNAs, even in the presence of inhibitors, with sensitivity and precision (2, 15).
Analyzing GE with ddPCR is targeted to provide traits of gene functions. Nowadays,
ddPCR is a powerful novel tool in GE analysis because it has advantages over other PCRs
when comparing sensitivity, precision, reproducibility, and quantification. Most studies
applying ddPCR to GE have been used for cancer and tuberculosis studies (15).
Other uses
Next-generation sequencing (NGS)

ddPCR quantifies NGS sample library preparations to increase sequencing accuracy and
reduce repeat cycles. Confirm sequencing results such as single nucleotide polymorphisms
or copy number variations with absolute quantification. ddPCR and NGS can provide a
better and more accurate quantitative measure of the fraction of gene mutant alleles in the
characterization of reference material (16).

NGS over ddPCR can identify cancer biomarkers more easily than other molecular tools.
NGS can find cancer DNA or identify mutations occurring in any place of the target genes
at a frequency as low as one or several thousand copies (17).

Single cell analysis

Cell-to-cell gene expression and genetic material variation among homogeneous post-
mitotic, parent, and stem cell types demand analysis from single cells. ddPCR needs low
copy number quantification to analyze a cell (18). This novel tool provides new chances to
undertake drug resistance studies based on individual cells to develop new therapies.
Scientists have developed an assay in which single-cell cytotoxicity was produced with the
generation of libraries of optically-coded droplets using ddPCR for anti-cancer drug
screening (19).
Genome edit detection

Gene edition techniques are becoming most used for modifying genetic material in
organisms. ddPCR provides a fast, accurate, and cost-profit assessment of NHEJ (Non-
homologous end joining) and HDR (Homology-directed repair) generated by CRISPR-
Cas9 or other types of gene editing tools (20, 21).

Discussion

It is clear PCR have allowed to amplify DNA to look for portraits on it for decades. Now,
have appeared a new PCR (ddPCR) with novel characteristics which enable discover new
outcomes in research related with genes. What is more, ddPCR has advantages over other
types of gene-amplification tools such as it needs only a few amounts of the sample to run
the process (2). ddPCR has multifield application in general it can be used for studies in
humans, animals, plants and microorganisms.

Even, ddPCR can cover those fields which time before were difficult to develop researches.
For instance, in the medicine field, ddPCR is useful to diagnose many pathologies, in this
way it can chose a better treatment for patients. Even it can be used in first death-cause in
the world (cardiovascular disease). This molecular tool

Nowadays, especially in developing countries, the use of ddPCR is quite expensive because
is an unknown tool which is used in a few laboratories only and its benefits are unknown in
the research area especially.

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Annexes

Table 1: Comparison among different type of PCRs

PCR Type Basic PCR qPCR rt-PCR ddPCR

Overview Analyze the Measures PCR Allows the use of Measures the fraction of
amount of amplification while it RNA as a template. adverse micro reactions
accumulated is working The RNA is to determine a specific
product at the reverse transcribed amount of copies.
end of the into
whole process complementary
DNA, using
reverse
transcriptase
Application Sequencing Gene expression Analysis of small Whole viral load
quantification genetic samples quantification
Genotyping Microarray Used in the Whole quantification of
verification diagnosis and nucleic acid standards.
quantification of
RNA virus
infections
Cloning Quality control and Analysis of mRNA Whole quantification of
assay validation transcripts next-gene sequencing
Pathogen detection (translocations) libraries
SNP genotyping Detection of Rare allele detection
expressed genes
Copy number Examination of Whole quantification of
variation transcript variants gene expression
Copy number Generation of
variation cDNA templates
for cloning and
MicroRNA Analysis sequencing.
Viral quantitation
siRNA/RNAi
experiments
Advantages/ Poor precision Expand the dynamic Requires intact No house-keeping gene
disadvantages Low range of detection mRNA Precision can be got by
sensitivity increasing the whole number
 Low resolution No processing post- The final product is of PCR replicates
PCR not subject to
RNase degradation
Post-PCR Detection is capable High complexity More tolerant to PCR
of a 2-fold change and problems inhibitors
processing
associated with its
sensitivity,
reproducibility, and
specificity
Short dynamic Collect data in the Quantitative as Capable of analyzing
range < 2 logs exponential growth well as qualitative abstract mixtures
phase of PCR analysis
Size-based The fluorescent No post-PCR Linear response to the
discrimination report signal is processing number of copies
only
directly proportional Simple, easy to present allows for minor
to the number of use, rapid and cost- fold change differences
amplicons generated effective to be detected
Results are not The cleaved probe High specificity
expressed as provides a permanent and the sensitivity
numbers
record amplification
of an amplicon
Quantitative - +++ +++ ++++

Table 2: Applications of ddPCR in different fields

Article Source studied Field

Digital PCR for Quantifying Circulating Cells Cardiovascular
MicroRNAs in Acute Myocardial Infarction and Disease
Cardiovascular Disease (22)
Developing multiplex ddPCR assays for SARS- SARS-CoV-2 Virology
CoV-2 detection based on probe mix and
amplitude based multiplexing (23)
Droplet digital PCR applications in the tuberculosis Mycobacterium Bacteriology
world (24) Tuberculosis
Reliable and robust droplet digital PCR (ddPCR) Mouse Assays
and RT-ddPCR protocols for mouse studies (25)
Application of Reverse Transcriptase PCR (RT- Escherichia coli Bacteriology
PCR) for rapid detection of viable Escherichia
coli in drinking water samples (26)
Sensitive and accurate quantification of human Plasmodium Parasitology
malaria parasites using droplet digital PCR
(ddPCR) (27)
Combining ddPCR and environmental DNA to Microorganisms Water
improve detection capabilities of a critically
 endangered freshwater invertebrate
Droplet digital polymerase chain reaction (ddPCR) Animals Food
assays integrated with an internal control for
quantification of bovine, porcine, chicken and
turkey species in food and feed (28)
Quantification of mitochondrial DNA copy number Cells Cancer
in suspected cancer patients by a well optimized
ddPCR method (29)
Droplet Digital PCR for Absolute Quantification of Plants pathogens Plants
Plant Pathogens (30)
Development of a Droplet Digital PCR for Sheep Veterinary
Detection of Trichuriasis in Sheep (31)
Assessing the Success of CRISPR Gene Therapies Genes Gene editing
Using ddPCR (32)

Figure 1: Steps of process of ddPCR

a) Sa
e) Water-in-oil droplet partitions